Cartilage/bone inducing materials for reparation

ABSTRACT

A cartrilage and bone morphogenetic repairing composition comprising a collagen-free aqueous solution of a polyoxyethylene-polyoxypropylene and an effective amount of a bone morphogenetic protein, the molecular weight of polyoxypropylene as a constituent of said polyoxyethylene-polyoxypropylene molecular is 900 to 4,000 in a unit of Dalton (D) and the ethylene oxide content is 5 to 90% by weight of the polyoxyethylene-propoxypropylene molecule whereby the solution is liquid at 1 to 30° C. and gelatinizes at about 37° C. and a method of using the same.

FIELD OF THE INVENTION

The present invention relates to a cartilage and bone morphogeneticrepairing composition for the treatment of bone fracture and bonedefect. In more detail, this invention is concerned with the cartilageand bone morphogenetic repairing composition which contains apolyoxyethylene-polyoxypropylene glycol and a bone morphogeneticprotein.

BACKGROUND OF THE INVENTION

For repairing cartilages and bones, in addition to autoplasty, there hasbeen practiced a procedure in which a prosthetic material for defectedsites of cartilage and bone composed of a combination of a bonemorphogenetic protein and a suitable carrier was imbedded in thedefected site. In practicing this, the defected site can be exposed onsurgical operation to apply a cartilage and bone repairing compositioncontaining a bone morphogenetic protein directly to the defected site,and thus, the materials in a solid form such as blocks, sponges, sheetsand the like which are easy to handle have been widely applied. Those ina semisolid form such as gels or pastes can also be used. As thecarriers which made such solid or semisolid forms applicable, there havebeen utilized, for example, metals such as stainless or titanium alloysor collagen and hydroxyapatite (HAP) or a mixture thereof.

On the other hand, an attempt has been made to administer a bonemorphogenetic protein for the treatment of bone fracture orosteoarthritis without requiring any surgical operation. Thisadministration mode has been earnestly desired from a viewpoint thatnon-invasive administration, namely, injection mode, would alleviatepains from patients. However, the injection route of a simple aqueousliquid preparation of a bone morphogenetic protein causes diffusion anddisappearance of the drug after administration, and so in order toachieve an effective administration, the bone morphogenetic proteinshould be retained in the injected site over a certain period of time.In view of the above, there has been envisaged a carrier which may be ina liquid state capable of passing through a needle on administration andthen phasetransited to a gel-like state after administration to retainthe bone morphogenetic protein in the injected site. Preferably, thecarrier may have non-toxicity, a good bio-compatibility and a highbio-absorption in a living body.

Collagen is a known carrier for a bone morphogenetic protein and isconfirmed to possess favorable bio-compatibility and bio-absorption(Japanese Patent Publication No. 75425/1993). Collagen with aninjectable character has also been reported, which may provide aninjectable cartilage and bone morphogenetic material (Japanese PatentPublication Nos. 23322/1995 and 53140/1993). However, collagen nowavailable for the use of medicines is derived from natural sources suchas cattle or a pig, so that its properties such as a molecular weight,an amino acid composition and a moisture holding property are not alwaysconstant. In addition, it has some side-effects such as antigenicitybecause it is a heterologous protein to humans. In particular,antigenicity cannot be completely eliminated even when atelocollagen;i.e., collagen from which teropeptide sites are removed, is used (J.American Academy of Dermatology 10, 638-646 and 647-651, 1984 and ibid,21 1203-1208, 1989).

On the other hand, it was reported that biodegradable polymers such aspolylactic acid or polylactic acid-glycolic acid copolymers can be usedas pharmaceutical carriers (U.S. Pat. No. 5,385,887 and Japanese PatentPublication No. 22570/1994). However, the biodegradable polymers are ina solid or semisolid state which may maintain a given form, and in viewof this, they are classified as a group of applicable materials tosurgical operation. Even if an injectable complex can be prepared usingsuch biodegradable polymers, an organic solvent should be employedduring the preparation process, which may easily anticipate the problemof inactivation of the active ingredient, a bone morphogenetic protein.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of this invention to provide a cartilage and bonemorphogenetic repairing composition, which can overcome the prior artdisadvantages or drawbacks as discussed above, which have a highbio-absorption and a good affinity to the active ingredient or a bonemorphogenetic protein, and which show the sustained disposition of abone morphogenetic protein by causing a temperature dependent sol-gelreversible transition with less side-effects such as antigenicity and soon.

The present inventors have made earnest studies on the relationshipbetween the active ingredient, a bone morphogenetic protein, and acarrier therefor in the case of a bone repairing method without surgicaloperation and have found that a certain class ofpolyoxyethylene-polyoxypropylene glycols can show a high bio-absorption,a good affinity to a bone morphogenetic protein and temperaturedependent sol-gel reversible transition. The present inventors haveprepared a bone morphogenetic composition by mixing an aqueouspolyoxyethylene-polyoxypropylene glycol solution and a bonemorphogenetic protein, which is an injectable liquid at a temperature offrom 1° C. to 30° C. at the time of administration and may begelatinized at around 37° C. within 3 minutes after administration. Ithas been found that ectopic cartilage and bone morphogenesis areaccomplished by administering said composition to mice intramuscularlyat the femoral muscle and then retaining a bone morphogenetic protein atthe administration sites in vivo upon which this invention has beencompleted.

This invention is concerned with a cartilage and bone morphogeneticrepairing composition which contains a polyoxyethylene-polyoxypropyleneglycol and a bone morphogenetic protein.

The polyoxyethylene-polyoxypropylene glycol(s) as used herein is ageneric name of nonionic surface active agents of a polymer type havingless hydrophilic polyoxypropylene as a hydrophobic group and ethyleneoxide as a hydrophilic group. It may be feasible to prepare surfaceactive agents having various properties by changing a molecular weightof the polyoxypropylene and a mixing ratio thereof to the ethyleneoxide. The synthesizable polyoxyethylene-polyoxypropylene glycols have amolecular weight of the polyoxypropylene in the range of 900-4,000 and apercent by weight of the ethylene oxide in the total molecule of 5% to90%. For instance, the polyoxyethylene-polyoxypropylene glycol blockpolymers (ADEKA®) manufactured by Asahi Denka Kogyo K.K. aresystematically named according to a molecular weight of polyoxypropyleneand a weight ratio of the ethylene oxide to be added and theclassification list thereof is shown in FIG. 1.

Industrial utilization of polyoxyethylene-polyoxypropylene glycolsincludes aperients, ointment bases, artificial blood, coating fortablets, excipients, solubilizers or solubilizing agents for injectionsand others in the field of pharmaceutics, in addition to the use asgeneral cleaning agents or antifoamings. In particular, Pluronic F-68 (amolecular weight of polyoxypropylene of 1,750 and an ethylene oxidecontent of 80%) has a remarkable antihemolytic action and has beenmarketed in the name of EXOCORPOL® from the Green Cross Corporation(polyoxyethylene-polyoxypropylene glycol) as an additive forextracorporeal circulation of blood. It is apparent from the results oftoxicity tests using various animals thatpolyoxyethylene-polyoxypropylene glycols have extremely low toxicity andlow irritative property, with no reports on possible side-effects suchas antigenicity and so on (Fragrance Journal, 7, 82-87, 1974). Theresults of toxicity tests are shown in Table 1.

TABLE 1 Results of acute toxicity tests using ADEKA ® Pluronics ADEKA ®Pluronics Animal Species LD₅₀(g/kg) L-44, L-62, L-64 Rats 5 F-68Mice >15 F-68 Rats, Rabbits, Dogs No acute toxicity P-85 Rats 34.6

Polyoxyethylene-polyoxypropylene glycols are superior in terms ofhandiness to collagen showing non-reversible phase-transition by changesin temperatures in the point that they show reversible sol-gelphase-transition. This property may be controlled by selection of theoptimum polyethylene-polyoxypropylene glycol for the temperature todevelop the phase-transition and by changing the concentration of anaqueous solution of said polyoxyethylene-polyoxypropylene glycol (Int.J. Pharm. 22, 207-218, 1984 and EP 0551626A1).

It is obvious from the foregoing that polyoxyethylene-polyoxypropyleneglycols have a superior nature as a drug carrier. Attempts have alreadybeen made to combine them with a low molecular weight drug such as localanesthetics, anticancer agents and so on (Int. J. Pharm. 8, 89-99, 1981and Chem. Pharm. Bull. 32, 4205-4208, 1984) and to admix with a highmolecular weight physiologically active protein such as interleukins andthe like (Pharm. Res. 9, 425-434, 1992).

This invention relates to a cartilage and bone morphogenetic repairingcomposition which contains a polyoxyethylene-polyoxypropylene glycol anda bone morphogenetic protein, wherein the polyoxypropylene as aconstituent of said polyoxyethylene-polyoxypropylene glycol has amolecular weight of about 1,500-4,000 and an ethylene oxide content ofabout 40-80%/molecule. Within the above ranges, there will be providedthe Pluronics capable of performing temperature-dependent sol-gelreversible transition, which characterized the present Pluronics.

Moreover, this invention relates to a cartilage and bone morphogeneticrepairing composition wherein a concentration ofpolyoxyethylene-polyoxypropylene glycols as described above in anaqueous solution is about 10-50%. It is known that the reversible phasetransition temperature of polyoxyethylene-polyoxypropylene glycolsvaries in general depending on the concentration of their preparedaqueous solutions, and the polyoxyethylene-polyoxypropylene glycolswithin the above-mentioned constituent ranges may gelate at around bodytemperature, i.e. about 37° C. at a concentration of about 10-90% in itsaqueous solution. As the most preferable example, there is prepared thepolyoxyethylene-polyoxypropylene glycol block polymer aqueous solutionof 15-30% concentration having a molecular weight of polyoxypropylene of3,850 and an ethylene oxide content of 70% (Pluronic F-127).

The bone morphogenetic protein (BMP) as used herein is the proteinhaving an activity to induce undifferentiated mesenchymal cells tocartilage cells, thereby performing bone morphogenesis.

The bone morphogenetic proteins used in this invention include, but arenot limited to, a series of proteins belonging to the TGF-β genesuperfamily such as BMP-2 to BMP-9 and so on, the protein named MP52,the protein named GDF-5 and the like. Particularly preferable BMP-2 is aprotein produced using Chinese hamster ovary (CHO) cells according tothe genetic engineering technology reported by Wang, et al. (Proc. Natl.Acad. Sci. USA, 87, 2220-2224, 1990 and U.S. Pat. No. 4,877,864), andparticularly preferable MP52 is a new protein produced according to thegenetic engineering technology suggested by the present inventors (ourcopending Japanese Patent Application Serial No. 531,621 filed Oct. 20,1977). This new protein can be produced by constructing a plasmidcontaining the DNA sequence coding the amino acid sequence as shown inSEQ ID NO.:2 of the Sequence Listing derived from MP52 described in saidJapanese patent application and having added the codon coding methionineat the N-terminal of said DNA sequence; transforming the plasmid intoE.coli; incubating the E.coli to obtain an inclusion body; andsolubilizing and purifying the inclusion body to obtain a monomerprotein, which is then dimerized and purified.

An aqueous solution of 15-30% polyoxyethylene-polyoxypropylene glycolblock polymer containing as an active ingredient BMP-2 or MP52 wasintramuscularly injected to mice at the femoral muscle. MP52 wasretained at the administered sites and then an ectopic cartilage andbone morphogenesis ability was observed in vivo.

There has not yet been reported to date an injectable cartilage and bonemorphogenetic repairing composition comprising apolyoxyethylene-polyoxypropylene glycol in combination with a bonemorphogenetic protein which may be useful for repair of cartilage andbone, especially as a treating agent for bone fracture.

The present invention is further concerned with a cartilage and bonerepairing composition containing a polyoxyethylene-polyoxypropyleneglycol and a bone morphogenetic protein.

Moreover, the present invention is concerned with a method of treatmentfor cartilage and bone repairing, by which a cartilage and bonemorphogenetic repairing composition comprising apolyoxyethylene-polyoxypropylene glycol in combination with a bonemorphogenetic protein is administered locally to the site of bonefracture or bone defect of human or animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a classification figure for AKEDA® Pluronics, wherein anethylene oxide content in terms of % by weight in a total molecule of apolyoxyethylene-polyoxypropylene glycol is indicated on the abscissa,while a molecular weight of the component polyoxypropylene in apolyoxyethylene-polyoxypropylene glycol is indicated on the ordinate.

FIG. 2 is soft X-ray photographs of the bone/cartilage calcified tissuesof the femur in the right hind leg of the mouse as obtained by Example4. The photographs (a) and (b) were taken after 2 weeks from theadministration of AKEDA® Pluronic F-127 solely and AKEDA® Pluronic F-127containing MP52, respectively. The apparently blackened parts in themuscle indicate ectopically formed bones.

FIGS. 3 a and 3 b are microscopic photographs of the stained tissues ofthe non-decalcified sections of the femur of the right hind leg of themouse as obtained by Example 4. Formations of bone matrices and bonematrices together with osteoblasts and of bone marrows can be confirmedby von-Kossa staining (a) and Hematoxylin-Eosin staining (b),respectively.

FIG. 4 is a plasmid map of the expression vector of the protein MP52 asobtained by Reference Example 1 (2).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The effects of this invention will be illustratively explained by way ofthe following Examples and Reference Examples. However, this inventionis not to be restricted by these Examples.

EXAMPLE 1

Preparation of Cartilage and Bone Morphogenetic Repairing CompositionContaining BMP-2

ADEKA® Pluronic F-127 (Asahi Denka Kogyo K.K.) is known to be one of theleast toxic polyoxyethylene-polyoxypropylene glycols (“SEIYAKU KOJO” 6,875-880, 1986). In 7.0 g of distilled water for injection was dissolvedunder ice-cooling 3.0 g of ADEKA® Pluronic F-127 to prepare a 30%aqueous solution of ADEKA® Pluronic F-127. The aqueous solution ofADEKA® Pluronic F-127 was poured potionwise under ice-cooling to a96-well titer plate at 360 μl/well, 40 μl of 0.01 N HCl containing 80 μgof BMP-2 was added to each well and mixed. The mixture was sterilized bypassing through a 0.22 μm filter at 4° C. to form a BMP-2 injection of atotal volume of about 400 μl (a final concentration of ADEKA® PluronicF-127 of 27%). Similarly, the BMP-2 injections having finalconcentrations of ADEKA® Pluronic F-127 of 10, 15, 18 and 22.5% wereprepared.

It was found that injection was feasible at 5° C. or lower in the caseof the final concentration of ADEKA® Pluronic F-127 of 27%, at 10° C. orlower in the case of the final concentration of ADEKA® Pluronic F-127 of22.5%, or at 25° C. or lower in the case of the final concentration ofADEKA® Pluronic F-127 of 10%-18%, while the ADEKA® Pluronic F-127injection phase-transited to a gel-like state at 37° C. was apreparation having a final concentration of 15% or higher. Accordingly,the most preferable is a preparation of ADEKA® Pluronic F-127 with thefinal concentration of 18%, which was in a liquid state at roomtemperature and showed a gelatinized state at 37° C.

EXAMPLE 2

Preparation of Cartilage and Bone Morphogenetic Repairing CompositionContaining MP52

The MP52 injections having final concentrations of ADEKA® Pluronic F-127of 10, 15, 18, 22.5 and 27% were prepared according to the sameprocedure as described in Example 1. The same injectable preparations asdescribed for the case of the BMP-2 was obtained according to MP52; thatis to say, the injectable preparations applicable at 5° C. or lower inthe case of the final concentration of ADEKA® Pluronic F-127 of 27%, at10° C. or lower in the case of the final concentration of ADEKA®Pluronic F-127 of 22.5%, or at 25° C. or lower in the case of the finalconcentration of ADEKA® Pluronic F-127 of 10%-18%.

EXAMPLE 3

Residual Rates of MP52 in vivo after Administration of Cartilage andBone Morphogenetic Repairing Composition

The ¹²⁵I-labeled MP52 injections having the final concentrations ofADEKA® Pluronic F-127 of 18, 22.5 and 27%, which had been preparedfollowing the same method and formulation as Example 2 except that¹²⁵I-labeled MP52 was further added, were intramuscularly administeredto male mice (ICR strain, 8 weeks old) under anesthesia at the femur ofthe right hind leg at 100 μl using a 23G needle (about 37 KBq¹²⁵I-MP52/site) and then the radioactivity in the right hind leg wascounted at 0.5, 2 and 8 hours after administration. The injection of an¹²⁵I-MP52 aqueous solution was used as a control. The results are shownin Table 2.

TABLE 2 ¹²⁵I-MP52 residual rates at the right hind leg afteradministration of ADEKA ® Pluronic preparations (ADEKA ® Pluronic F-127final concentration of 18%) or aqueous liquid preparation Time (hr)Pluronic Preparation Aqueous Liquid Preparation 0.5 60.5% 32.7% 2 19.7%13.8% 8 14.9%  7.9%

It was clearly shown in Table 2 that MP52 whenpolyoxyethylene-polyoxypropylene glycols were used as a pharmaceuticalcarrier could apparently be retained more as compared with the casewhere a simple MP52 aqueous solution was injected. Also similar resultswere obtained using the injection of Example 1.

EXAMPLE 4

Pharmacological Effect on Ectopic Cartilage and Bone Morphogenesis

The MP52 injection of ADEKA® Pluronic F-127 final concentration of 18%as prepared in Example 2 was intramuscularly administered to male mice(ICR strain, 8 weeks old) under anesthesia at the femur of the righthind leg at 100 μl using a 23G needle (20 μl MP52/site). The ADEKA®Pluronic F-127 injection containing no MP52 was used as a control.Cartilage and bone formation was determined after two weeks from theadministration. The mice were sacrificed by vertebral cervicaldislocation and the right hind leg of the administration site was cutoff and bone formation at the administration site was examined by usinga soft X-ray irradiator. The results are shown in FIG. 2 (n=5). Asapparent from the soft X-ray images, no shadow was observed in themuscles at the administration site in the case of ADEKA® Pluronic F-127only (FIG. 2- a), while clear shadow was observed in 80% or more of theanimals with ADEKA® Pluronic F-127 containing MP52 (FIG. 2- b).

And then, after taking images using soft X-ray, the specimens were keptin 10% formalin and histologic examination was carried out. Themicroscopic photograph of the stained tissue of the mouse seen at theright end in FIG. 2- b is shown in FIG. 3. In FIG. 3, deposition ofcalcium was observed at the shadowed portion by von-Kossa staining (FIG.3- a) and osteoblasts, bone matrices and bone marrows were confirmed byHematoxylin-Eosin staining (FIG. 3- b), whereby bone formation wasconfirmed. No inflammatory reaction was observed. In these figures, bonematrix, osteoblast and bone marrow are abbreviated as BM, OB and MA,respectively.

Similar test was carried out using the BMP-2 injection having an 18%final concentration of ADEKA® Pluronic F-127 to give similar results.

From the aforesaid results, safety and usefulness of apolyoxyethylene-polyoxypropylene glycol were confirmed when used as acarrier for the bone forming factor.

Referential Example

Production of New Protein MP52

1. Construction of Vector

(1) Isolation of Variant MP52 Mature Part

Human MP52cDNA was amplified by polymerization chain reaction (PCR) ofthe mature part only, using the plasmid vector containing cDNA describedin WO93/16099 (pSK52s) as a template DNA.

A part of the DNA of the mature type MP52 gene was substituted accordingto the method for increasing in the productivity of the desired proteinby increasing the AT content around the initiation codon ATG (reportedby M. Nobuhara et al., Agric. Biol. Chem., 52 (6), 1331-1338, 1988).

Substitution was carried out according to the PCR method using anorthodromic PCR primer of SEQ ID NO.:3. The DNA sequence of the PCRprimer utilized the DNA described in SEQ ID.:3 as an orthodromic primerand that described in SEQ ID No.:4 as an antidromic primer. SequenceNo.: 3 and No.:4 are those described in the Nobuhara et al reference.

PCR was carried out by adding in the same test tube the template DNA (10ng), 50 picomoles each of the orthodromic and antidromic PCR primers,dNTP (0.2 mmol) and MgCl₂ (1.5 mmol), together with Taq DNA polymerase(5 U).

The PCR of 30 cycles was performed, each cycle comprising denaturation(94° C., one minute) and primer annealing (55° C., one minute) andprimer elongation (72° C., 2 minutes). (All the following PCRs wereperformed under the above-defined conditions.)

The product from the PCR method was separated by electrophoresis in 1.5%low-melting agarose (available from FMC) to cut out the DNA composed ofabout 360 bp corresponding to the amino acid sequence of SEQ ID NO.:2,which is defined as Fragment 1.

(2) Construction of E. coli Expression Vector for the Present Protein

In order to increase the replication number of plasmid, the replicationorigin was altered from pBR cell line to pUC cell line. The tac promoterregion of commercially available E. coli expression vector pKK223-3(purchased from Pharmacia Biotech AB) was digested by the restrictionenzymes SspI and EcoRI, treated with Mung Bean Nuclease (Takara ShuzoK.K., Catalogue No. 2420A), ligated to the initiation codon site ofFragment 1 with T4DNA Ligase (Takara Shuzo K.K., Catalogue No. 2011A),and the rrnBT₁T₂ terminator region of pKK223-3 was digested with therestriction enzymes SalI and SspI, ligated to the termination codon siteof Fragment 1 digested with SalI, integrated into the SmaI site of pUC18to construct the expression vector for the production of the presentprotein [pKOT245 (FIG. 4)] which was deposited (Accession NumberBikokenki FERM-P P-14895) at the National Institute of Bioscience andHuman-Technology (NIBH), Agency of Industrial Science and Technologylocated in 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan on Apr.14, 1995, and transferred to a deposit (Accession No. BIKOKEN-KIBP-5499) on Apr. 10, 1996 according to Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms. The DNA ofpKOT245 has a length of 3.7 kb. The expression vector for the presentprotein as constructed was determined for its base sequence by means ofPharmacia ALF DNA sequencer.

(3) Transformation

Transformation was performed according to the rubidium chloride methodby Kushner et al. (Genetic Engineering, p. 17, Elsevier (1978)). That isto say, pKOT245 was migrated into a host E. coli W3110M according to theabove-mentioned procedure to prepare the E. coli capable of producingthe present protein.

2. Cultivation

(1) Cultivation

The present protein producing E. coli was precultured in modified SOCmedium (Bacto tryptone 20 g/l, Bacto yeast extract 5 g/l, NaCl 0.5 g/l,MgCl₂.6H₂O 2.03 g/l, Glucose 3.6 g/l) and 100 ml of the myceliumsuspension was added to 5 L of the productive medium (Bacto tryptone 5g/l, Citric acid 4.3 g/l, K₂HPO₄ 4.675 g/l, KH₂PO₄ 1.275 g/l, NaCl 0.865g/l, FeSO₄.7H₂O 100 mg/l, CuSO₄.5H₂O) 1 mg/l, MnSO₄.nH₂O 0.5 mg/l,CaCl₂.2H₂O 2 mg/l, Na₂B₄O₇.10H₂O 0.225 mg/l, (NH₄)6/6Mo₇O₂₄.4H₂O 0.1mg/l, ZnSO₄.7H₂O 2.25 mg/l, CoCl₂.6H₂O 6 mg/l, MgSO₄.7H₂O 2.2 g/l,Thiamine HCl 5.0 mg/l, Glucose 3 g/l) and then cultured with stirringand aeration in a 10 L culture tank andisopropyl-β-D-thiogalactopyranoside was added at a concentration of 1 mMat the stage of a logarithmic growth phase (OD₅₅₀=5.0) and thencultivation was continued until the OD₅₅₀ reached 150. During thecultivation, the temperature was controlled to 32° C. and a pH value wasadjusted to 7.15 by adding ammonia, while a dissolved oxygenconcentration was controlled to 50% of air saturation by increasing astirring speed to prevent any reduction in the dissolved oxygenconcentration. On the other hand, cultivation was carried out by addinga 50% glucose solution at 0.2% concentrations using as a standard arapid increase in the dissolved oxygen concentration in order to keep ahigh mycelium concentration.

(2) Preparation of E. coli Inclusion Body

The cultured broth obtained as above was centrifuged to recover themycelium, which was then suspended in 25 mM Tris-HCl buffer containing10 mM etylenediaminetetraacetic acid (pH 7.3) and then bacteria werebroken by means of a mycelium breaking apparatus (available from GohlinCo., Inc.) and centrifuged again to recover the precipitate containingthe inclusion body.

3. Purification

(1) Solubilization of E. coli Inclusion Body

The E coli inclusion body was washed thrice with 1% Triton X-100 andthen centrifuged at 3,000×g at 4° C. for 30 minutes. The precipitatethus obtained was solubilized under ultasonification with 20 mM Tris-HClbuffer, pH 8.3, 8 M urea, 10 mM DTT and 1 mM EDTA.

(2) Purification of Monomer

The solubilized liquid thus obtained was centrifuged at 20,000×g at 4°C. for 30 minutes to recover the supernatant. The resultant supernatantwas passed through SP-Sepharose FF (Pharmacia) which had beenequilibrated with 20 mM Tris-HCl buffer (pH 8.3), 6 M urea, and 1 mMEDTA, washed with said solution and then eluted with said solutioncontaining 0.5 M sodium chloride. To the eluate were added Na₂SO₃ andNa₂S₄O₆ at the respective final concentrations of 111 mM and 13 mM andsulfonation was carried out at 4° C. for 15 hours. The sulfonatedsolution was gel-filtrated with Sephacryl S-200 (Pharmacia) which hadbeen equilibrated with 20 mM Tris-HCl buffer (pH 8.3), 6M urea, 0.2 Msodium chloride and 1 mM EDTA to obtain a single sulfonated proteinmonomer of the invention.

(3) Refolding

To a solution of the sulfonated protein monomer of the invention wasadded a 9 times volume of 50 mM Na-Glycine buffer (pH 9.8), 0.2 M sodiumchloride, 16 mM CHAPS, 5 mM EDTA and 2 mM GSH (glutathione of reducedtype) and 1 mM GSSG (glutathione of oxidized type), and then the mixturewas stirred at 4° C. for one day to perform refolding.

(4) Purification of Dimer

The sample was diluted into a two-times volume of purified water andthen added by 6 N NaCl adjusting pH to approximately pH 7.4 and placedto isoelectric precipitation. The precipitation collected bycontrifugation at 3,000×g for 20 minutes was solubilized in a solutionwith 30% acetonitrile containing 0.1% TFA. The solution was diluted intoa two-times volume of purified water and loaded on RESOURCE RPC column(Pharmacia) of a reverse-phase HPLC which had been equilibrated with 25%acetonitrile containing 0.05% TFA, and then eluted with a lineargradient of 25-45% acetonitrile containing 0.05% TFA. The eluate wasmonitored at 280 nm absorbance. The purified homodimer protein fractionswere collected and lyophilized by Speedback Concentrator (Servant Co.).

(5) Determination of Physico-chemical Properties of the Present PurifiedProtein

(a) Analysis of N-terminal Amino Acid Sequence

The present purified protein obtained as above was analyzed for theN-terminal amino acid sequence by means of an amino acid sequencer,Model 476A (Applied Biosystems) to confirm the amino acid sequence fromthe N-terminal up to the 30^(th) amino acid.

(b) Analysis of Amino Acid Composition

The present purified protein obtained as above was investigated by meansof amino acid analyzer [PICO TAG System (Waters Co., Ltd.)]. The resultsare shown in Table 3 wherein the numerical indication means the numberof the amino acid residue per monomer.

TABLE 3 AminoAcid Practical No. Estimated No. Asx 11.5 12 Glx 10.9 11Ser 8.4 9 Gly 4.3 4 His 4.0 4 Arg 7.7 7 Thr 5.4 6 Ala 7.3 7 Pro 10.2 10Tyr 2.9 3 Val 5.7 7 Met 5.1 4 ½Cys 2.6 7 Ile 4.9 6 Leu 10.0 10 Phe 4.0 4Lys 5.9 6 Trp — 2 Sequence Length 119(c) Analysis by Electrophoresis

The molecular weight of the present purified protein obtained above wasconfirmed by means of SDS-PAGE under non-reductive conditions to show amolecular weight of about 28KDa.

It has been proven from the results shown in the aforesaid items (a),(b) and (c) that the present protein is a protein consisting of 119amino acid residues simply starting from the N-terminal of Pro.

Industrial Utilization

The cartilage and bone morphogenetic repairing composition according tothe invention can be applied to the affected site in the bone fracturetherapy requiring no surgical operation in a simple and painless mannerdue to a high bio-absorption, a favorable affinity to the activeingredient, i.e., a bone morphogenetic protein, and a temperaturedependent sol-gel reversible transition. Thus, the drug effect of a bonemorphogenetic protein may be sustained and further a cartilage and bonemorphogenetic repairing composition with less side-effects may beprovided.

1. A cartilage and bone morphogenetic repairing composition comprising acollagen-free aqueous solution of a polyoxyethylene-polyoxyporpyleneglycol and an effective amount of a bone morphogenetic protein, whereinthe molecular weight of polyoxypropylene as a constituent of saidpolyoxyethylene-polyoxypropylene glycol molecule is 900 to 4,000 in aunit of Dalton (D) and the ethylene oxide content is 5 to 90% by weightof the polyoxyethylene-polyoxypropylene glycol molecule whereby thesolution is liquid at 1 to 30° C. and gelatinizes at about 37° C.
 2. Thecartilage and bone morphogenetic repairing composition as claimed inclaim 1, wherein said bone morphogenetic protein is BMP-2.
 3. Thecartilage and bone morphogenetic repairing composition as claimed inclaim 1, wherein said bone morphogenetic protein is MP52.
 4. Thecartilage and bone morphogenetic repairing composition as claimed inclaim 1, wherein the polyoxypropylene as a constituent of saidpolyoxyethylene-polyoxypropylene glycol has a molecular weight of about1,500-4,000 in a unit of Dalton (D) and the ethylene oxide content isabout 40-80% per molecule.
 5. The cartilage and bone morphogeneticrepairing composition as claimed in claim 4, wherein a concentration ofsaid polyoxyethylene-polyoxypropylene glycol in an aqueous solution isabout 10-50%.
 6. A method of repairing a cartilage and bone fracture ina warm-blooded animal comprising administering locally to thewarm-blooded animal a composition of claim 1 at the site of a bone orcartilage fracture for a time and under conditions of repairingcartilage and bone.
 7. The method of claim 6 wherein the bonemorphogenetic protein is BMP-2.
 8. The method of claim 6 wherein thebone morphogenetic protein is MP52.
 9. The method of claim 6 wherein thepolyoxypropylene as a constituent of thepolyoxyethylene-polyoxypropylene glycol of said composition has amolecular weight of about 1,500 to 4,000 in a unit of Dalton (D) and theethyleneoxide content of the polyoxyethylene-polyoxypropylene glycol isabout 40 to 80% per molecule.
 10. The method of claim 9 wherein thepolyoxyethylene-polyoxypropylene glycol is about 10 to 50% by weight ofthe aqueous solution.